Regulation of Steroid Hormone Production by Inter-Organelle Substrate Exchange

描述（由申请人提供）：皮质醇等类固醇激素是多种生物过程的关键调节剂，这些生物过程通过与转录因子核受体超家族的成员结合而引起生理变化。由于糖皮质激素控制许多基因的表达，因此使用多种机制来确保严格控制激素的产生。我们的初步研究结果为促肾上腺皮质激素（ACTH）刺激的微管依赖性线粒体运输在维持最佳糖皮质激素输出中的作用提供了证据。我们还鉴定了几种蛋白质，包括 GTPase RhoA 和 Rho 效应器 diaphanous 1 (DIAPH1)，它们可以促进这种运动。 ACTH 信号传导控制 DIAPH1 与 RhoA 和其他几个结合伙伴相互作用的能力。然而，这些相互作用的功能意义尚未完全阐明，我们也没有定义 ACTH 刺激的线粒体运动是否通过促进 ER 和线粒体之间的底物传递来促进皮质醇的产生。我们假设 ACTH 刺激的线粒体运输速率增加是细胞器间底物交换和糖皮质激素生物合成所必需的。为了检验这一假设，我们将定义 ACTH 信号传导控制 DIAPH1 复合物组装的机制（具体目标 1）。我们已经生成了表达针对 DIAPH1 的 shRNA 的稳定细胞系，以定义该蛋白质在线粒体运动和激素产生中的作用。我们还将评估 DIAPH1 与几种结合配偶体之间相互作用的功能意义，包括微管蛋白、驱动蛋白和氧甾醇结合蛋白相关蛋白 2 (ORP2)。还将进行研究以确定磷酸化在控制 DIAPH1 功能中的作用（具体目标 2）。我们发现 ACTH 信号传导刺激 DIAPH1 的磷酸化。将使用磷酸突变体和磷酸特异性抗体来检查这种翻译后修饰对 DIAPH1 功能的作用。最后，我们将确定 11-脱氧皮质醇在 ER 和线粒体之间转运的机制（具体目标 3）。我们发现 ORP2 与 DIAPH1 共纯化，并假设这种脂质结合蛋白可能促进 ER 和线粒体之间的底物转移。我们将描述 ORP2 在皮质醇生物合成中的作用，并确定 ER 和线粒体之间的融合是否在细胞器间底物传递中发挥作用。从这些研究中获得的知识将为肾上腺皮质中类固醇激素合成的一个很大程度上未被探索的方面提供有价值的信息。我们的研究结果还可以深入了解其他代谢过程中内质网和线粒体之间底物转移的机制。

DESCRIPTION (provided by applicant): Steroid hormones such as cortisol are key regulators of a diverse array of biological processes that evoke physiological changes by binding to members of the nuclear receptor super family of transcription factors. Because glucocorticoids control the expression of numerous genes, multiple mechanisms are used to ensure tight control over hormone production. Our preliminary findings provide evidence for the role of adrenocorticotropin (ACTH)-stimulated, microtubule-dependent mitochondrial trafficking in maintaining optimal glucocorticoid output. We have also identified several proteins, including the GTPase RhoA and the Rho effector diaphanous 1 (DIAPH1), that act to facilitate this movement. ACTH signaling controls the ability of DIAPH1 to interact with RhoA and several other binding partners. However, the functional significance of these interactions has not fully been elucidated, nor have we defined if ACTH-stimulated mitochondrial movement enables cortisol production by facilitating the delivery of substrate between the ER and mitochondria. We hypothesize that ACTH-stimulated increases in the rate of mitochondrial trafficking are required for inter- organelle substrate exchange and glucocorticoid biosynthesis. To test this hypothesis we will define the mechanism by which ACTH signaling controls DIAPH1 complex assembly (Specific Aim 1). We have generated a stable cell line that expresses shRNA against DIAPH1 to define the role of the protein in mitochondrial movement and hormone production. We will also assess the functional significance of the interactions between DIAPH1 and several binding partners, including tubulin, kinesin, and the oxysterol- binding-protein related protein 2 (ORP2). Studies will also be performed to define the role of phosphorylation in controlling DIAPH1 function (Specific Aim 2). We have found that ACTH signaling stimulates the phosphorylaton of DIAPH1. The role of this post-translational modification on DIAPH1 function will be examined using phospho-mutants and a phospho-specific antibody. Finally, we will determine the mechanism by which 11-deoxycortisol is transported between the ER and mitochondria (Specific Aim 3). We have found that ORP2 co-purifies with DIAPH1 and hypothesize that this lipid binding protein may facilitate substrate transfer between the ER and mitochondria. We will characterize the role of ORP2 in cortisol biosynthesis and also determine if fusion between ER and mitochondria play a role in inter-organelle substrate delivery. The knowledge gained from these studies will provide valuable information about a largely unexplored facet of steroid hormone synthesis in the adrenal cortex. Our findings may also provide insight into the mechanism by which substrates are transferred between the ER and mitochondria in other metabolic processes.